Cycle sequencing polymerase chain reaction (PCR) products has proven to be an effective alternative to the more traditional M13 sequencing technique. Advantages include the use of thermostable polymerases that allow high annealing and extension temperatures (Gyllenstein and Erlich, Proc. Natl. Acad. Sci. USA 85:7652 (1988), Innis et al, Proc. Natl. Acad. Sci. USA 85:9436 (1988)), as well as the ability to directly utilize double-stranded DNA for templates (Carothers et al, Biotechniques 7:494; Sears et al, Biotechniques 13:626 (1992)). High temperature annealing and extension reduce ambiguous sequencing data that arise because of mispriming and/or secondary structure of the template. The capability of sequencing double-stranded DNA allows PCR products to be sequenced immediately after amplification. However, a simpler method for sequencing PCR products is to incorporate sequence delimiters directly into the PCR amplification process.
A sequencing method based on the incorporation of 5'-.alpha.-thiotriphosphates into PCR products has been demonstrated (Nakamaye et al, Nucl. Acids Res. 16:9947 (1988)). 5'-.alpha.-Thiotriphosphates were incorporated into DNA during PCR amplification and the positions of incorporated base-specific 5'-.alpha.-thiotriphosphates were revealed by chemical degradation with either 2-iodoethanol or 2,3-epoxy-1-propanol. Also 5'-.alpha.-thiotriphosphates have been used to sequence single stranded M13 DNA (Labeit et al, Meth. Enzymol. 155:166 (1987)). After incorporation by Klenow into primer extension products, the positions of the 5'-.alpha.-thiotriphosphates were revealed by exonuclease III digestion. However, attempts to combine the best features of both methods (PCR amplification and enzymatic digestion) to reveal the sequence delimiters have proven unsatisfactory because of uneven band intensity (Nakamaye et al, Nucl. Acids Res. 16:9947 (1988); Olsen and Eckstein, Nucl. Acids Res. 17:9613 (1989)). The present invention overcomes the problems of the art and provides a simple and accurate method of amplifying and sequencing nucleic acids in a single step. Furthermore, the method is fast and amenable to automation.